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PHARMACOLOGICAL REVIEWS Vol. 61, No. 3
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 620/3517435
Pharmacol Rev 61:358 –372, 2009 Printed in U.S.A.

Therapeutic Targeting of Nuclear Protein Import in

Pathological Cell Conditions
MIRNA N. CHAHINE AND GRANT N. PIERCE

Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Department of Physiology, Faculties of Medicine and
Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
II. Nucleocytoplasmic trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
A. Classic nuclear import cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
B. Alternative nuclear import pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
III. Dysregulation of nuclear protein import: evidence for its involvement in pathological cell
conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
A. Dysfunctional cargo trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
B. Transport receptors and the transport driving force . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
1. Transport receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

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2. Transport-driving force. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
C. Nuclear pore complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
IV. Targeting nuclear import as a strategy to alter disease progression. . . . . . . . . . . . . . . . . . . . . . . . . . . 365
A. Pharmacological modulation of nuclear import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
1. Targeting the nuclear pore complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
2. Targeting nuclear import via transport receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
3. Targeting nuclear localization of key transcription factors in disease. . . . . . . . . . . . . . . . . . . . 365
a. Targeting nuclear factor-␬B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
b. Targeting nuclear factor of activated T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
4. Targeting nuclear import with kinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
B. Improving therapeutic delivery of DNA to the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
V. Applications for therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
A. Viral therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
B. Cancer therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

Abstract——Proteins enter the nucleus through
the nuclear pore complex. Once in the nucleus, some
proteins, such as transcriptional regulators, can
turn genes on or off, and change the composition of
the cell and its function to meet the demands of its
environment. This process of protein import into the
nucleus is highly controlled and regulated by the
expression or function of single cargoes, transport
receptors, or the transport channels themselves.
Thus, these components of the import process have
Address correspondence to: Dr. Grant N. Pierce, Institute of Car-
diovascular Sciences, St. Boniface General Hospital Research Cen-
tre, 351 Tache Avenue, Winnipeg, Manitoba, Canada, R2H 2A6.
E-mail: gpierce@sbrc.ca
This article is available online at http://pharmrev.aspetjournals.org.
doi:10.1124/pr.108.000620.
358
an impact on transport capacity, which subsequently
affects gene expression, signal transduction, and cell
growth and development. With such a key position in
the process of cell growth, it is reasonable to hypoth-
esize that alterations in nuclear protein transport may
play an important role in pathological cell conditions
that have abnormal cell growth as a central feature.
Indeed, there are now sufficient data to demonstrate
that alterations in nuclear protein transport partici-
pate in alterations in cell proliferation and hypertro-
phy. Further study is needed to provide definitive
proof that changes in nuclear protein import directly
participate in the pathogenesis of diseases such as
hypertension, atherosclerosis, cancer, viral infection,
and diabetes. However, the data to date have, on select
occasions, led to a clear association of alterations in
nuclear transport with disease states. Furthermore,
 THERAPEUTIC TARGETING OF NUCLEAR PROTEIN IMPORT IN DISEASE 359
this research has led to the important identification of lication, to improve drug delivery during cancer ther-
new targets within the process of nuclear protein im- apy, and, in general, to modify cell growth and
port that hold therapeutic promise to inhibit viral rep- viability during disease conditions.

I. Introduction lems. Targeting the nuclear protein machinery now con-

stitutes an approach used to avoid viral replication in
The movement of proteins from the cell cytoplasm into
the nucleus (used as antiviral therapies), improve drug
the nucleus can induce gene expression and alter cell
delivery (used in anticancer therapies such as chemo-
function, composition, and morphology. Nuclear protein
therapy or for drugs lacking cellular selectivity), and aid
import is essential, therefore, for cell growth. Many fac-
in nonviral gene transfer.
tors are important for nuclear transport. These include
targeting signals responsible for protein import [nuclear
localization signal (NLS1)] and protein export (nuclear II. Nucleocytoplasmic Trafficking
export signal), transport receptors called karyopherins
Access to the nucleus is limited by the nuclear enve-
(or the importin and exportin superfamilies), other pro-
lope, a double-membrane structure in which large pore
teins constituting the nuclear pore complex (NPC) in-
structures named nuclear pore complexes are embedded
cluding the nucleoporins (Nups) and Ran GTPase-acti-
(Poon and Jans, 2005). Mammalian NPC is composed of
vating protein (RanGAP) (Poon and Jans, 2005). The
30 to 50 Nups (Kau and Silver, 2004) that play precise
transport of molecules (⬎40 kDa) in or out of the nucleus
roles in NPC assembly and function (Terry et al., 2007).
consists of three phases: recognition by a receptor, dock-
Proteins smaller than 40 kDa can diffuse through the
ing, and transport through the NPC (Kau et al., 2004)
NPC, whereas larger proteins are actively transported
(Fig. 1). Each loading and unloading of cargoes and the
rate of transport through the NPC are highly controlled
by the Ran GTPase (Kau et al., 2004). Thus, the process
that participates in the nuclear import of key proteins,
such as transcription factors, DNA-binding proteins,
polymerases, and kinases, is responsible for regulating
not only gene expression but also larger processes, such
as cell proliferation and cell differentiation, phenomena
occurring during cell mitosis, and embryonic develop-
ment. Alterations in this highly regimented process
have been observed in a wide spectrum of pathological
cell conditions (e.g., cell hypertrophy, angiogenesis, and
a wide range of diseases including atherosclerosis, hy-
pertension, cancer, viral infection, and diabetes). This
review discusses the dysregulation of the nuclear pro-
tein import machinery that is highly associated with the
pathological states cited in section III and therapeutic
interventions recently developed to address these prob-

1
Abbreviations: CAS, cellular apoptosis susceptibility; CNI-H1194,
2-amino-4-(3,5-diacetylphenyl)amino-6-methylpyrimidine; cNLS, clas-
sical NLS; DTPA, diethylenetriamine pentaacetic acid; EBOV, Ebola
virus; EGF, epidermal growth factor; EGFR, epidermal growth factor
receptor; ERK, extracellular regulated kinase; HCMV, human cytomeg-
alovirus; HIV, human immunodeficiency virus; HSP70, 70-kDa heat
shock protein; IFN, interferon; I␬B, inhibitor of nuclear factor-␬B; LPC,
lysophosphatidylcholine; MAPK, mitogen-activated protein kinase;
NF-␬B, nuclear factor-␬B; NFAT, nuclear factor of activated T cells;
NLS, nuclear localization signal; NPC, nuclear pore complex; NPI,
nuclear protein import; Nups, nucleoporins; oxLDL, oxidized low-
density lipoprotein; PARP, poly(ADP-ribose polymerase); PBC, pri- FIG. 1. The nuclear protein import cycle. An NLS-containing cargo
mary biliary cirrhosis; PCNA, proliferating cell nuclear antigen; (molecules ⬎40 kDa) is recognized by importin-␣ that forms a het-
PD98059, 2⬘-amino-3⬘-methoxyflavone; PIC, preintegration complex; erodimer with importin-␤1. The trimeric complex translocates into the
PY-STAT1, tyrosine-phosphorylated signal transducer and activator nucleus through the NPC by docking of Nups. The release of the NLS
of transcription 1; RanGAP, Ran GTPase-activating protein; SB203580, cargo in the nucleus requires the binding of RanGTP to importin-␤
dissociating the complex. Importin-␣ is exported via its own export re-
4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole;
ceptor known as CAS. The complex RanGTP-importin-␤ is recycled to the
SN-50, NF-␬B cell-permeable inhibitory peptide (AAVALLPAVLLAL- cytoplasm and the RanGTPase-activating protein RanGAP stimulated
LAPVQRKRQKLMP); VSMC, vascular smooth muscle cell; WGA, hydrolysis of Ran GTP to RanGDP, thus releasing the importins for
wheat germ agglutinin. another transport cycle.
360 CHAHINE AND PIERCE

through the nuclear import machinery (Gasiorowski and

Dean, 2003).
The import of cargo proteins into the nucleus is medi-
ated by different types of NLSs. These NLSs are recog-
nized by members of the importin superfamily, of which
there are multiple ␣ and ␤ forms. Nuclear protein import
is mediated either by one of a number of importin-␤ or by
a heterodimer of importins ␣/␤1 (Fig. 1) if the proteins to
be imported contain classic NLSs (cNLSs) (Goldfarb et
al., 2004; Poon and Jans, 2005; Lange et al., 2007; Ratan
et al., 2008).
A. Classic Nuclear Import Cycle
During the classic nuclear protein import cycle, im-
portin-␣ plays the role of an adapter to bond cNLS car-
goes. The heterodimer of importins ␣/␤1 is translocated
into the nucleus through the NPC by docking to Nups
(Köhler et al., 1999; Ratan et al., 2008). In the nucleus,
the transport receptor importin-␤1 binds to the small
nuclear GTPase Ran-GTP, which induces a conforma-
tional change thus causing the dissociation of importin-␣
and the release of the cNLS cargo (Stewart, 2007; Ratan
et al., 2008). Although importin-␤1 returns rapidly to
the cytoplasm, importin ␣ is exported to the cytoplasm
via its nuclear export factor cellular apoptosis suscepti-
bility (CAS), which binds to importin ␣ in the presence of
RanGTP (Köhler et al., 1999). Hydrolysis of the GTP in
FIG. 2. The Ran cycle. RanGTP is hydrolyzed by the cytoplasmic Ran-
the cytoplasm releases importin-␣ for a new import cycle GAP into RanGDP, which is then transported into the nucleus by NTF-2.
(Ratan et al., 2008). In the nucleus, RanGEF catalyzes recharging of RanGDP with GTP,
which is then free to bind importins and mediate their translocation into
Note that the RanGTPase, a small GTPase, provides the cytoplasm through the NPC.
the required energy to control transport in each direc-
tion through two forms of Ran: RanGDP at high levels in
the cytoplasm and RanGTP at high levels in the nucleus decades. For example, changes in the expression, struc-
(Gasiorowski and Dean, 2003; Lange et al., 2007). As ture, and/or function of ion channels will produce signifi-
described above, the gradient of RanGTP/RanGDP cant effects on tissue function and viability (Mukherjee
across the nuclear envelope constitutes the driving force and Spinale, 1998; Mukherjee et al., 1998). Modification
for protein trafficking through the NPC (Gasiorowski of the function of molecules that interact with ion chan-
and Dean, 2003) (Fig. 2). nels can also modulate tissue function (Pitt, 2007). Thus,
these channels represent an important therapeutic tar-
B. Alternative Nuclear Import Pathways get to minimize the clinical effects of a disease. The
It is also important to note that there are alternative concept that changes in nucleocytoplasmic trafficking
nuclear protein import pathways using other carriers through the nuclear pore may be involved in disease
and NLSs from those observed in the classic nuclear processes is generally the same. Changes in the expres-
import pathway. This allows the import of ribosomal sion, structure, and/or function of the pore represent a
proteins, histones, and heterogeneous nuclear ribo- critical juncture to modify cell morphology, function,
nucleoproteins (Stewart, 2007). Importin- and Ran-inde- growth, proliferation, and even cell death. These effects
pendent nuclear import pathways have also been re- may be achieved in three basic ways:
ported for the import of molecules such as ␤-catenin, 1. The transport of a specific cargo is affected when
importin-␤1, calmodulin, ERK, importin-␣, and STAT changes occur at the level of that cargo.
family proteins (Poon and Jans, 2005; Sorokin et al., 2. The transport of all cargoes recognized by a specific
2007). receptor is affected when modifications occur at the
level of that transport receptor.
III. Dysregulation of Nuclear Protein Import: 3. The transport process as a whole can be affected
Evidence for Its Involvement in Pathological when alterations occur at the level of the NPC
Cell Conditions itself. This may have the most dramatic effects on
Channels found in the plasma membrane of a cell have protein transport into the nucleus (Terry et al.,
been associated with organ function and dysfunction for 2007).
 THERAPEUTIC TARGETING OF NUCLEAR PROTEIN IMPORT IN DISEASE 361

Although this field is relatively young, evidence is A. Dysfunctional Cargo Trafficking

emerging that all of the processes involved in the move-
ment of signaling proteins into the nucleus may be al-
tered during disease and may participate in the patho-
logical characteristics of that disease. The nuclear
accumulation of proteins ultimately represents an im-
balance of both the import and export processes. Even
though this review will focus on the effects occurring
during nuclear import only, it is important to remember
that signaling proteins are normally in constant move-
ment in and out of the nucleus in a balance between the
rates of import and export (Gama-Carvalho and Carmo-
Fonseca, 2001). This review article focuses on the poten-
tial for this balance to be disturbed during a disease
process (Table 1).
Alterations in the regulatory mechanisms for nucleo-
cytoplasmic trafficking of cargo proteins (essentially the
movement of transcriptional factors from the cytoplasm
into the nucleus) could result in the localization of these
proteins in the wrong subcellular compartments and,
subsequently, pathological cell states. Several proteins
have been associated with a variety of diseases through
an abnormal nuclear localization. For example, the tran-
scriptional activator nuclear factor ␬B (NF-␬B) has a
predominant nuclear localization in breast, ovary, colon,
pancreas, and thyroid tumor cells (Brand et al., 1996;
Rayet and Gélinas, 1999; Huang et al., 2000; Lee et al.,
2007), and in vascular smooth muscle cells, endothelial

TABLE 1
Dysregulation of nuclear protein import and associated pathological cell conditions
Protein Pathological States

Target cargo protein
Transport receptors and the
transport-driving force
Nuclear pore complex
Activation and nuclear mislocalization of NF-␬B Tumors of breast, ovary, colon, pancreas, thyroid
Atherosclerotic plaques
Activation and nuclear mislocalization of HIF-1␣ Breast and prostate cancers,
hepatocarcinogenesis
Activation and nuclear mislocalization of NFAT Malignant transformation, cancer development,
tumor invasion, and metastasis
Diabetes
Nuclear mislocalization of cdk2, cd4, cyclin B1, Cell proliferation associated with atherosclerosis
cyclin D1 in cells exposed to oxLDL
Nuclear mislocalization of PCNA and phospho- Cell proliferation associated with atherosclerosis
ERK in cells exposed to oxLDL (short exposure)
Nuclear mislocalization of PCNA in cells exposed Cell proliferation associated with hypertension
to stretch
Nuclear mislocalization of phospho-p38 in cells Cell apoptosis associated with atherosclerosis
exposed to oxLDL (long exposure) and to
ceramide
Aberrant expression in importins ␣1␣2 Defects in fertility and embryogenesis
Truncated form of importin-␣ lacking NLS-binding Breast cancer cell lines
domain
Importin-␣1 overexpression Breast cancer, melanoma patients
Karyopherin ␣1 sequestrated in the cytoplasm by Ebola virus infection
EBOV protein
Nuclear mis-localization of importin-␤ in cells Cell stress
exposed to hydrogen peroxide
Nuclear mislocalization of importin-␣ in cells Cell stress
exposed to oxidative
Cytoplasmic mislocalization of CAS in cells Cell stress
exposed to oxidative
Cytoplasmic mislocalization of CAS in cells Cell stress
exposed to ceramide
CAS overexpression Tumors and cancer cells (colon cancer, breast
cancer, and liver neoplasms)
Cytoplasmic mislocalization of Ran/TC4 in cells Cell stress
exposed to hydrogen peroxide
Overactivation of RanGAP in cells exposed to LPC Atherosclerosis and cancer
mNup98 down-regulation Influenza virus
Nup 153 and p62 degradation Poliovirus and rhinovirus
Nup214/Nup88 complex up-regulation Acute myelogenous leukemia
Nup88 overexpression Tumor cell lines, primary human solid tumors
Autoantibodies against gp210 protein and Nup Primary biliary cirrhosis
p62
Nup 153 degradation induced by hydrogen Cell stress
peroxide
p62 and Nup 153 upregulation Cell stretch associated with hypertension
p62 up-regulation induced by oxLDL (short Cell proliferation associated with atherosclerosis
exposure)
p62 down-regulation induced by oxLDL (long Cell apoptosis associated with atherosclerosis
exposure)
Nup155 mutation Atrial fibrillation and early sudden cardiac
death
p62 mutation Infantile bilateral striatal necrosis
Neuroendocrinological disease (triple A
syndrome)
362 CHAHINE AND PIERCE
cells, and macrophages of atherosclerotic plaques (Brand
et al., 1996). The molecular mechanism leading to NF-␬B
activation and its subsequent import into the nucleus is
very well described. Stimuli, such as inflammatory cyto-
kines, reactive oxygen intermediates, and microorganisms,
induce the phosphorylation of NF-␬B inhibitor (I␬B) by the
I␬B kinase complex. This allows the unmasking of the
NF-␬B NLS and the activation of NF-␬B followed by its
nuclear import (Kau and Silver, 2003; Kau et al., 2004).
Abnormal localization of NF-␬B in the nucleus could be
due to an alteration in I␬B activity, a hyperactivation of
the upstream kinases resulting in I␬B phosphorylation
and degradation, or an improper acetylation by p300
(Rayet and Gélinas, 1999; Chen and Greene, 2003; Kau et
al., 2004).
Cell cycle protein regulators (cdk2, cd4, cyclin B1, and
cyclin D1) and proliferating cell nuclear antigen (PCNA)
translocate into the nucleus to modulate the cell cycle.
Oxidized low-density lipoprotein (oxLDL) is thought to
be involved in the accelerated cell proliferation and cell
death associated with atherosclerosis. oxLDL can induce
cell proliferation through, in part, affecting the nuclear
import of cell cycle proteins (Zettler et al., 2003). Low
concentrations of oxLDL or short exposure times of aor-
tic vascular smooth muscle cells (VSMCs) to oxLDL can
induce cell proliferation (Zettler et al., 2003; Chahine et
al., 2009). On the other hand, the nuclear translocation
of cell cycle proteins is inhibited by higher oxLDL con-
centrations or longer exposure times (Zettler et al., 2004;
Chahine et al., 2009). VSMCs exposed to ceramide re-
spond with a decrease in PCNA and cyclin A transloca-
tion into the nucleus (Faustino et al., 2008). Ceramide
blocks cell proliferation and induces cell apoptosis. Nu-
clear transport, therefore, may represent the mecha-
nism through which oxLDL and ceramide regulate the
degree of cell proliferation (Chahine et al., 2009) or cell
apoptosis (Faustino et al., 2008; Chahine et al., 2009)
that are relevant to the atherosclerotic process.
Regulatory molecules are not the only factors that have
the capacity to alter cell growth and nuclear protein im-
port. When cells were exposed to mechanical factors such
as cell stretch, cell growth (hyperplasia and hypertrophy)
was stimulated (Richard et al., 2007). At the same time,
nuclear protein import was increased in these cells com-
pared with control cells. Continuous, intermittent cell
stretch is an important component of vascular hyperten-
sion. The changes in nuclear protein import, therefore,
represent a mechanism to explain the accelerated growth
of the arterial wall in hypertension (Richard et al., 2007)
and a checkpoint that could be targeted to block this
response.
B. Transport Receptors and the Transport Driving
Force
Many pathological cell conditions or diseases are char-
acterized by a mutation or an altered expression of the
nuclear protein import receptors (such as importin-␣ or
-␤) or a disruption of the RanGTP/GDP gradient (called
the transport-driving force) (Terry et al., 2007).
1. Transport Receptors. Importin-␣ binds to cNLS-
containing proteins and links them to importin-␤, the
karyopherin that ferries the ternary complex through
the NPC. Importin-␣ is exported from the nucleus back
to the cytoplasm by CAS protein (Goldfarb et al., 2004;
Kodiha et al., 2008). Because importins play a major role
in the regulation of gametogenesis and early embryogen-
esis (Terry et al., 2007; Ratan et al., 2008), it is under-
standable that aberrant expression patterns for the im-
portins ␣1 and ␣2 would result in an altered import of
cNLS cargoes and, consequently, defects in fertility and
embryogenesis as demonstrated in Drosophila melano-
gaster (Goldfarb et al., 2004; Terry et al., 2007).
Alterations in importin-␣ have been reported during
cancer. A truncated form of importin-␣ lacking the NLS
binding domain has been discovered in breast cancer cell
lines (Kim et al., 2000; Kau et al., 2004). Overexpression
of importin-␣1 has been also found in breast cancer
(Dahl et al., 2006). BRCA1 is a nuclear protein trans-
ported into the nucleus by the karyopherin pathway
(Thakur et al., 1997) possibly linking importin-␣1 ex-
pression with breast carcinogenesis (Dahl et al., 2006).
Importin-␣1 overexpression has also been closely asso-
ciated with poor survival and prognosis in patients with
melanoma (Winnepenninckx et al., 2006).
During viral pathogenesis, abnormalities in trans-
porter receptor trafficking have been observed (Reid
et al., 2006). During Ebola virus (EBOV) infection, for
example, the virus is able to resist antiviral drugs
such as interferon (IFN)-␣/␤ or IFN-␥ by inhibition of
the IFN-␣/␤ or IFN-␥ signaling (Harcourt et al., 1999;
Reid et al., 2006). One of the major steps in this signal-
ing pathway is the nuclear import of protein tyrosine-
phosphorylated signal transducer and activator of tran-
scription 1 (PY-STAT1). Reid and coworkers (2006) showed
that infection of cells with EBOV blocks the IFN-induced
accumulation of PY-STAT1. They reported that, to inhibit
this signaling pathway, the protein EBOV VP 24 seques-
trates karyopherin-␣1, the previously identified NLS re-
ceptor for STAT1 (Sekimoto et al., 1997; McBride et al.,
2002; Melen et al., 2003), in the cytoplasm. This, in turn,
alters the interaction of karyopherin-␣1 with the NLS of
PY-STAT1 leading to inhibition of the nuclear import of
this protein, a key step in both IFN-␣/␤ or IFN- ␥ signaling
(Reid et al., 2006).
Cell stress is an essential component of the patho-
physiology of ischemia, heart failure, hypertension, dia-
betes, and cancer. It has been reported that in response
to cell stress (such as UV irradiation, oxidative stress,
and heat shock stress), transport receptors such as im-
portins are mislocalized (Kodiha et al., 2004; Miyamoto
et al., 2004). For example, importin-␤ has been localized
in the nucleus of cells exposed to hydrogen peroxide
(Kodiha et al., 2004). Importin-␣ has been localized in
 THERAPEUTIC TARGETING OF NUCLEAR PROTEIN IMPORT IN DISEASE
the nucleus of cells exposed to oxidative stress (Kodiha
et al., 2008) and to all cellular stresses cited above
(Miyamoto et al., 2004).
Nuclear accumulation of importin-␣ may be due to an
increase in nuclear import of CAS or a decrease in the
nuclear export of CAS (Kodiha et al., 2008). Because
CAS recycles importin-␣ to the cytoplasm, any change in
its localization will effectively alter nuclear trafficking
in both directions. Exposure of VSMCs to ceramide in-
duced a decrease in the nuclear localization of CAS
through p38 mitogen-activated protein kinase (MAPK)
activation (Faustino et al., 2008). This is thought to
decrease the recycling of importin-␣, thereby depressing
the nuclear import of proliferative genes such as PCNA
and cyclin A (Faustino et al., 2008). As discussed in
section III.A, ceramide is an important second messen-
ger that stimulates apoptosis and also induces a signif-
icant antiproliferative effect on VSMCs (Faustino et al.,
2008). On the other hand, CAS overexpression has been
reported in several different tumors and cancer cells
(colon cancer, breast cancer, and liver neoplasms)
(Brinkmann et al., 1996; Behrens et al., 2001; Wellmann
et al., 2001; Kau et al., 2004). CAS overexpression was
suggested to accelerate the recycling of importin-␣ to the
cytoplasm which enhanced the nuclear import of cargo
proteins such as p53, BRCA1, and RB (Behrens et al.,
2003; Kau et al., 2004) that can induce cell proliferation
or prevent cell apoptosis (Kau et al., 2004; Poon and
Jans, 2005; Terry et al., 2007).
2. Transport-Driving Force. In several pathological
cell conditions, particularly during oxidative stress, ab-
normalities in the enzymes controlling the Ran gradient,
such as RanGTPase, have been observed (Czubryt et al.,
2000; Kodiha et al., 2004; Miyamoto et al., 2004). Expo-
sure of VSMCs to hydrogen peroxide has been found to
increase the RanGTPase (Ran/TC4, a small Ras-related
GTPase) in the cytosol, thereby decreasing nuclear im-
port (Czubryt et al., 2000). These results have been
confirmed in a subsequent work by use of the same
oxidative stress (Miyamoto et al., 2004) and HeLa cells
(Kodiha et al., 2004).
During atherosclerosis and in cancer, an important
mitogenic second messenger is lysophosphatidylcholine
(LPC) (Chai et al., 1996; Fang et al., 2000; Faustino et
al., 2007b). Exposure of VSMCs to LPC stimulates
ERK1/2 activity, which in turn increases RanGAP activ-
ity (ERK1/2 shares docking sequences with RanGAP).
This induces the hydrolysis of Ran-GTP leading to
higher levels of RanGDP in the cytosol, which causes an
increase in RanGTP in the nucleus, enabling more im-
portins (␣ and ␤) to be recycled to the cytoplasm, which
increases nuclear import (Faustino et al., 2007b). In
conclusion, it is clear that oxidants are potential triggers
for changing the ratio of RanGTP/RanGDP, thereby al-
tering nuclear protein import.
363
C. Nuclear Pore Complex
Changing NPC composition or expression levels could
markedly alter nuclear transport and subsequently cel-
lular function. Increasing the number of channels may
increase transport characteristics. Furthermore, the NPC
is made up of a number of nuclear pore proteins called
nucleoporins. It is possible that any alteration in the Nups
could indirectly or directly lead to a large spectrum of
diseases.
Viral infection through DNA and some RNA viruses,
including poliovirus, rhinovirus, and influenza, can each
target a subset of Nups for degradation or selective
inhibition. For example, in 293T and Madin-Darby
canine kidney cells, influenza virus down-regulates
mNup98, a nucleoporin that is a docking site for mRNA
export factors. This disrupts the mRNA nuclear export
machinery rendering the cells highly permissive to in-
fluenza virus replication (Satterly et al., 2007). This
viral effect is in direct competition with interferon-␥-
mediated up-regulation of mNup98 (Enninga et al.,
2002; Satterly et al., 2007; Terry et al., 2007) and con-
stitutes a viral strategy to promote viral replication
(Satterly et al., 2007). Poliovirus (Gustin and Sarnow,
2001) and rhinovirus (Gustin and Sarnow, 2002) induce
both Nup 153 and p62 degradation. This degradation
has functional consequences. Nup153 interacts with Ran
GDP, and the protein p62 is found in the central channel of
the NPC (Guan et al., 1995). The protein p62 has been
shown to interact with importin-␤ family members (Hu et
al., 1996; Moroianu et al., 1996; Bonifaci et al., 1997; Ya-
seen and Blobel, 1997; Kehlenbach et al., 1999; Petersen et
al., 2001; Gustin and Sarnow, 2002) and NTF2 (Paschal
and Gerace, 1995), which is a critical protein for the proper
recycling of Ran between the nucleus and cytoplasm (Rib-
beck et al., 1998; Smith et al., 1998; Gustin and Sarnow,
2002). This might explain why an inhibition of nuclear
import correlated well with the degradation of Nup153 and
p62 in cells infected with poliovirus or rhinovirus (Gustin
and Sarnow, 2002). The inhibition of nuclear import by the
virus may represent a novel strategy to evade host im-
mune defenses by preventing signal transduction into the
nucleus (Gustin and Sarnow, 2001, 2002).
Several cancer-associated fusion proteins have been
identified involving Nups. In patients with acute myelog-
enous leukemia, chromosomal rearrangements can result
in the fusion of Nups, such as Nup98 or Nup214 (or CAN)
with HOXA9 (a homeobox transcription factor) or DEK (a
DNA-binding protein), respectively (Kau et al., 2004; Poon
and Jans, 2005). Up-regulation of the Nup214/Nup88 com-
plex has been observed during the development of leuke-
mia (Shu et al., 2008). Nup88 was overexpressed in a series
of tumor cell lines and primary human solid tumors (Gould
et al., 2002; Agudo et al., 2004; Zhang et al., 2007; Shu et
al., 2008). The fusion of Nups prevents them from being
incorporated into the NPC, thus retaining them in the
364 CHAHINE AND PIERCE

cytoplasm, and impairing their binding to transport recep-

tors (Kau et al., 2004; Faustino et al., 2007a).
Liver diseases have been associated with changes in
Nups (Wesierska-Gadek et al., 2008). Primary biliary
cirrhosis (PBC) is a chronic, progressive, cholestatic au-
toimmune liver disease characterized by destruction of
intrahepatic bile ducts, portal inflammation, and devel-
opment of cirrhosis and hepatic failure (Nakamura et
al., 2007). PBC targets two major Nups: gp210 and p62
(Wesierska-Gadek et al., 1995, 2007, 2008). Further-
more, autoantibodies against Nups have also been de-
tected in ⬃30% of patients with PBC (Invernizzi et al.,
2005; Nakamura et al., 2007; Wesierska-Gadek et al.,
2008). Because the levels of PBC-specific anti-NPC an-
tibodies correlate with the severity of disease, this may
be of important prognostic value (Invernizzi et al., 2001,
2005; Wesierska-Gadek et al., 2006, 2008). Indeed, the
presence of anti-gp210 antibodies has been identified as
a risk factor for the progression to end-stage hepatic
failure (Nakamura et al., 2007).
Oxidative cell stress may be a common factor inducing
an alteration in NPC composition or NPC permeability.
Oxidative stressors such as hydrogen peroxide induce a
degradation of Nup153 resulting in a decrease in nu-
clear import through a mechanism independent of the
ERK1/2 MAPK pathway (Kodiha et al., 2004). Oxidant
treatment also induced the import of Nup 153 and 88 FIG. 3. Effects of different cellular stressors on nuclear protein import
into the nucleus where they become components of high (NPI), NPC density, and cell growth through MAPK activation. LPC
(Faustino et al., 2007b), mechanical stretch (Richard et al., 2007) and
molecular complexes. Importin-␣ is sequestered within short exposure of VSMC to oxLDL (6 h) (Chahine et al., 2009) induces a
these high molecular complexes in nuclei of stressed high level of activation of the ERK MAPK pathway, an up-regulation of
NPI and NPC expression (up-regulation of Nup p62), and a stimulation of
cells, thus inhibiting nuclear import (Kodiha et al., the cell cycle (PCNA). The increased import of specific proteins into the
2008). Oxidized compounds can also affect the NPC. nucleus (i.e., transcription factors) could lead directly to the activation of
Short-term exposure of VSMCs to oxLDL induced an genes that cause cell proliferation (left side). Conversely, ceramide
(Faustino et al., 2008) and longer exposure of VSMC to oxLDL (48 h)
increase in p62 expression (Chahine et al., 2009). (Chahine et al., 2009) induces increased levels of activation of the p38
ERK1/2 MAPK activity was directly involved in this MAPK pathway, and an inhibition of NPI and NPC expression (down-
regulation of Nup p62). The decreased import of specific proteins into the
up-regulation. Nuclear protein import and cell prolifer- nucleus (i.e., transcription factors) could directly induce the activation of
ation were similarly stimulated by this short exposure to genes that cause cell apoptosis (cleavage of PARP) (right side). Nuclear
oxLDL (Chahine et al., 2009). However, exposure of protein import and the technique of microinjection: a to e represent
confocal images of VSMC microinjected with Alexa488-BSA-NLS sub-
VSMCs to oxLDL for longer durations induced an in- strate contained in a micropipette into a single cell. Images were taken
crease in p38 MAPK activity resulting in the down- during (a) and after injecting (b– e) cells at different time points. a to c are
images of the same cell. c to e represent images at 30 min after injection;
regulation of p62 expression, a depressed rate of nuclear c, image of a control cell (untreated); d, image of a cell exposed to oxLDL
import and an induction of cell apoptosis (Chahine et al., for 6 h; e, image of a cell exposed to oxLDL for 48 h. The signal strength
2009) (Fig. 3). in the nucleus is proportional to the amount of substrate present in the
nucleus. The pseudocolor scale represents the intensity of fluorescence
A mechanical stimulus is also a mechanism to induce levels from 0 (black) to 255 nm (white). BSA, bovine serum albumin.
cell stress. Cell stretching will stimulate cell growth via
cell hyperplasia and hypertrophy (Richard et al., 2007).
Mechanical stretch induced an up-regulation of NPC fibrillation and early sudden cardiac death (Zhang et al.,
expression (two nucleoporins, Nup153 and p62). This 2008). They showed that the NUP155 mutation inhibited
was associated with an activation of the MAPK pathway HSP70 mRNA export and HSP70 protein nuclear import
(Markovics et al., 1974; Maul et al., 1980; Richard et al., (Zhang et al., 2008). The authors suggested that “NUP155
2007). It also correlated with an increase in nuclear may act as a key upstream gene that causes atrial fibril-
protein import (Richard et al., 2007) (Fig. 3). lation by regulating the expression of downstream genes
Changes in the NPC have been directly linked to the such as HSP70 by controlling mRNA nucleocytoplasmic
development of atrial fibrillation and early sudden cardiac export.” Thus, this study has provided a novel, ion channel-
death by Zhang et al. (2008). They used human genetic and independent mechanism for the pathogenesis of an electro-
mouse gene-targeting approaches to detect a mutation in physiological disturbance in the heart through an alter-
the gene encoding nucleoporin NUP155 that leads to atrial ation in the NPC (Zhang et al., 2008).
 THERAPEUTIC TARGETING OF NUCLEAR PROTEIN IMPORT IN DISEASE
Two other examples directly involving nuclear-traf-
ficking diseases of the central nervous system are both of
a nuclear pore complex protein causing a Mendelian
disease in humans. A mutation of the nucleoporin p62
protein has been suggested to cause autosomal reces-
sive, infantile bilateral striatal necrosis, which is a neu-
rodegenerative disorder (Basel-Vanagaite et al., 2006).
Their findings suggested that p62 has a cell-type-specific
role and is important in the degeneration of the basal
ganglia in humans (Basel-Vanagaite et al., 2006). The
triple A syndrome is an autosomal recessive neuroendo-
crinological disease in humans that exhibits defective
NPC targeting (Cronshaw and Matunis, 2003; Basel-
Vanagaite et al., 2006; Kiriyama et al., 2008). The caus-
ative mutations for this syndrome have been identified
in a gene that encodes ALADIN, a component of the
NPC. The primary defect caused by the mutant ALADIN
is the selective failure to import the DNA repair proteins
aprataxin and DNA ligase I into the nucleus, resulting
in increased DNA damage and subsequent cell death
under oxidant stress (Kiriyama et al., 2008).
IV. Targeting Nuclear Import as a Strategy to
Alter Disease Progression
The preceding discussion has provided evidence from
many independent studies identifying the association
and the involvement of nuclear trafficking with different
pathological states. Because this has occurred at three
different general sites within the process of nuclear pro-
tein import, it offers multiple therapeutic targets to at-
tempt to normalize nuclear trafficking as a strategy for
the treatment of diseases.
A. Pharmacological Modulation of Nuclear Import
1. Targeting the Nuclear Pore Complex. Several ap-
proaches can be used to modulate nuclear import. One of
them consists of targeting the nuclear import at the level of
the NPC (Gasiorowski and Dean, 2003) specifically by tar-
geting conserved FG or FXFG repeats of the NPC that bind
to the importin family members. Monoclonal antibodies
(including mAb414 and RL2) against the FG and FXFG
epitopes of the nucleoporins have been used successfully in
rat liver nuclear envelopes to block the translocation of
proteins through the NPC (Snow et al., 1987). Monoclonal
antibodies prevent cargo from associating around the rim
of an NPC, which ultimately inhibits its movement into the
nucleoplasm (Gasiorowski and Dean, 2003). Alternatively,
several Nups in higher organisms are characterized by the
covalent addition of a single O-linked N-acetylglucosamine
to serine and threonine residues (Gasiorowski and Dean,
2003). This sugar moiety can mediate some of the interac-
tions with importins. For example, wheat germ agglutinin
(WGA) is a lectin that binds to N-acetylglucosamine. When
added to isolated rat liver nuclei, WGA can inhibit nuclear
import by associating with the sugar-modified nucleopor-
ins and blocking the channel (Finlay et al., 1987; Adam
365
and Adam, 1994; Gasiorowski and Dean, 2003). An exam-
ple of the utility of this approach is provided during adeno-
viral delivery of DNA. In this process, the NPC serves as a
docking site for incoming virus particles, contains a parti-
cle dissociation activity, and provides a gateway for nu-
clear import of the uncoated viral DNA (Horwitz and Loeb,
1990; Greber et al., 1996, 1997). Inhibitors of O-linked
NPC glycoproteins, WGA and the RL1 antibody, prevent
stable virus docking at the nuclear envelope and also pre-
vent the disassembly of the capsid and ultimately inhibit
the nuclear import of the DNA-associated protein VII
(Greber et al., 1997).
It is important to note that the experiments cited above
were not performed on intact cells. The use of monoclonal
antibodies is problematic when it is necessary to target
intracellular proteins because of a lack of accessibility.
However, a new method for the intracellular delivery of
antibodies has been developed recently that uses a novel
hemagglutinating virus of Japan envelope vector system
(Kondo et al., 2008). The hemagglutinating virus of Japan
envelope is an inactivated Sendai virus particle that can
fuse to the external membrane of a cell and thereby deliver
a variety of molecules into living mammalian cells (Kondo
et al., 2008). This new method could hold great promise for
the future to deliver monoclonal antibodies that selectively
block the NPC.
In conclusion, there is still a very limited number of
compounds but no drugs to date that can selectively
target the NPC. WGA is limited in its use because it
nonspecifically blocks all proteins that are normally im-
ported through the NPC (Gasiorowski and Dean, 2003).
Thus, this inhibitor can only be useful clinically as a
diagnostic test for a protein potentially targeted to the
nucleus (Adam and Adam, 1994; Gasiorowski and Dean,
2003), or more generally to detect the presence of auto-
antibodies to correlate with the severity of disease. De-
spite its limitations, it may have important prognostic
value in autoimmune diseases, such as primary biliary
cirrhosis, in particular (Wesierska-Gadek et al., 2008).
2. Targeting Nuclear Import via Transport Recep-
tors. Two peptide inhibitors, bimax1 and bimax2, have
been identified recently as specific blockers of nuclear
import activity mediated by the classic nuclear import
pathway (importin-␣/␤ pathway) (Kosugi et al., 2008).
Both peptides bind tightly to importin-␣ independently
of importin-␤, which prevents the release of the cargo
into the nucleus. These peptides were designed without
use of protein structural information. Instead, an activ-
ity-based profile was generated for a linear peptide,
which represented the functional contribution of various
amino acids substituted at each position within a tem-
plate peptide sequence. This approach was successful in
obtaining peptides that bind with high specificity and
high affinity (Kosugi et al., 2008).
3. Targeting Nuclear Localization of Key Transcrip-
tion Factors in Disease. Drugs are normally used to
directly stimulate or, more frequently, inhibit the activ-
366 CHAHINE AND PIERCE

ity of a target protein. However, a drug could be just as

effective if it could induce an alteration in the function of
a target simply through a change in its localization.
Cargo proteins such as transcription factors are down-
regulated when localized to the cytoplasm, then once
activated they translocate into the nucleus. Thus, the
activity of these transcription factors can be regulated
by their subcellular localization. Small molecules that
alter the localization of transcription factors, for exam-
ple, can be used to control nuclear transport (Kau and
Silver, 2003; Kau et al., 2004).
a. Targeting Nuclear Factor-␬B. Inhibition of NF-␬B
suppresses carcinogenesis (Holmes-McNary and Bald-
win, 2000; Takada et al., 2004; Piva et al., 2005; Lee et
al., 2007). However, most of the inhibitors lack specific-
ity and inhibit other signaling pathways (Lee et al.,
2007). Different strategies have been developed for can-
cer therapy, including the use of cell-permeable pep-
tides. The cell-permeable peptide strategy specifically
targets the NF-␬B complex by blocking its nuclear local-
ization. For example, peptides such as SN-50 and o,o⬘-
bismyristoyl thiamine disulfide have been designed to
mimic the sequence of p50, which is responsible for
transporting the NF-␬B complex from the cytoplasm to
the nucleus. The peptides compete with NF-␬B com-
plexes, thus blocking NF-␬B nuclear import (Pieper and
Riaz-ul-Haq, 1997; Shoji et al., 1998; Lee et al., 2007). FIG. 4. Calcineurin/NFAT shuttling and an approach to abolish the
b. Targeting nuclear factor of activated T cells. In- deleterious effects of exaggerated NFAT transcriptional activity. Cal-
cineurin, translocated into the nucleus via importin-␤1, plays a crucial
volvement of the calcineurin/nuclear factor of activated role in the transcriptional activity of the calcineurin/NFAT signaling
T cells (NFAT) pathway is critical in many diseases. The cascade leading to myocardial hypertrophy. To inhibit the interaction
between calcineurin and importin-␤1 and, consequently, the harmful
conventional inhibitors for NFAT pathway are immu- effects of calcineurin/NFAT signaling, a synthetic NLS peptide has been
nosuppressive drugs such as CsA and FK506 (Zhu and designed to mimic the NLS sequence of calcineurin that is recognized by
McKeon, 1999). After entry into cells, CsA and FK506 form importin-␤1. This creates a competition, thus inhibiting the import of
calcineurin into the nucleus and abolishing the transcription of genes
complexes with their cellular partners and block the phos- important in myocardial hypertrophy (model proposed by Hallhuber et
phatase activity of calcineurin (Zhu and McKeon, 1999; al., 2006). ␤1, importin-␤1.
Hallhuber et al., 2006). However, because CsA and FK506
block the enzymatic activity of calcineurin, they do not
specifically target NFAT. Therefore, these immunosup- scription of genes important in myocardial hypertrophy
pressive agents have severe side effects (Kiani et al., 2000; and identifies a potentially novel therapeutic strategy to
Lu and Huan, 2007). The ultimate goal is a highly selective inhibit myocardial hypertrophy (Hallhuber et al., 2006).
inhibitor for NFAT signaling. More specific targeting has The same approach may be used in other diseases. For
been developed in myocardial hypertrophy in which the example, in diabetes, a slight increase in endogenous
calcineurin/NFAT signaling cascade is crucial. Calcineurin calcineurin/NFAT signaling may increase ␤-cell mass
is responsible for dephosphorylating NFAT in the cytosol, and improve function in patients with type 2 diabetes
thus enabling its nuclear import (Zhu and McKeon, 1999). mellitus (Heit et al., 2006; Heit, 2007). Future studies
Its presence in the nucleus is also significant in ensuring might test the use of small-molecule activators of NFAT
the full transcriptional activity of NFAT (Zhu and McKeon, proteins as novel therapeutics in the treatment of type 2
1999). In angiotensin II-stimulated hypertrophy, inhibi- diabetes mellitus (Crabtree and Olson, 2002; Heit,
tion of the calcineurin/importin-␤1 interaction by a syn- 2007).
thetic competitive peptide (KQECKIKYSERV), which 4. Targeting Nuclear Import with Kinase Inhibitors.
mimics the calcineurin NLS, subsequently prevented cal- Alterations in MAPK activation have important conse-
cineurin nuclear import (Fig. 4). The inhibition of cal- quences on the movement of proteins into the nucleus.
cineurin nuclear import by peptide competition for the Therefore, MAPK inhibitors have been used extensively
binding of the nuclear import protein importin-␤1 repre- in vitro and have a regulatory effect on uncontrolled cell
sents a sophisticated approach to abolishing the deleteri- growth and cell apoptosis through an action on nuclear
ous effects of exaggerated NFAT transcriptional activity protein import (Czubryt et al., 2000; Faustino et al.,
(Hallhuber et al., 2006). This results in a suppressed tran- 2007b; Richard et al., 2007; Chahine et al., 2009). For
 THERAPEUTIC TARGETING OF NUCLEAR PROTEIN IMPORT IN DISEASE
example, the ERK1/2 MAPK inhibitor PD98059 normal-
ized the increases in p62 expression levels, nuclear im-
port rate, and cell proliferation induced by a short ex-
posure of VSMCs to oxLDL (Chahine et al., 2009),
mechanical stretch (Richard et al., 2007), or LPC (Faustino
et al., 2007b). In addition, the p38 MAPK inhibitor
SB203580 normalized the decreases in p62 expression
levels, nuclear import rate, and cell number induced by
longer exposure of VSMCs to oxLDL (Chahine et al.,
2009) or to ceramide (Faustino et al., 2008). The mech-
anism responsible for the effects of the p38 inhibitor
involved CAS localization (Faustino et al., 2008). CAS
was delocalized from the nucleus after exposing cells to
ceramide and treatment with the p38 MAPK inhibitor
redistributed CAS to the nucleus, which allowed for the
progression of the transport cycle. This suggests that
CAS could be a target of p38 MAPK. CAS contains target
motifs to MAP/ERK kinase, a kinase upstream to p38
(Faustino et al., 2008). Taken together, these results
have shown that even when nuclear import is depressed,
it is possible to restore it by targeting the upstream
kinases instead of directly targeting the different com-
ponents of the transport machinery. Thus, these very
encouraging in vitro results have led to the use of kinase
inhibitors in clinical therapy. However, when applied as
a clinical therapy, kinase inhibitors lack cellular selec-
tivity. Because they are mostly small lipophilic mole-
cules, their distribution throughout the body would be
widespread, exposing both diseased tissues and healthy
tissues to the drug. Thus, they can be toxic, particularly
with more chronic treatments (Kerkelä et al., 2006;
Force et al., 2007; Temming et al., 2008). A drug delivery
approach for cell-type-specific kinase inhibitors might be
a better strategy (Temming et al., 2008). The first step
consisted of the recognition of the receptor present on
diseased cells by the kinase inhibitor-carrier conjugate,
followed by their binding, thus leading to receptor inter-
nalization (Temming et al., 2008). This intracellular de-
livery approach ensures that the coupled drug acts spe-
cifically in diseased cells, whereas normal cells are not
affected by the drug. These approaches may be very
useful tools for improving drug delivery specifically into
tumor cells, angiogenic endothelial cells, or fibrotic he-
patic and renal cells (Temming et al., 2008). By using
this strategy, a kinase inhibitor can specifically interact
with the cytoplasmic kinases of diseased cells that sub-
sequently can regulate nuclear import.
B. Improving Therapeutic Delivery of DNA to the
Nucleus
Peptides resembling NLS sequences can be used to
facilitate nuclear uptake of exogenous DNA (van der Aa
et al., 2006). Another new approach has used histone to
mediate DNA delivery. Histones contain protein trans-
duction domains that enable them to enter an intact cell
in a receptor- and energy-independent manner (Wag-
staff et al., 2007). Very recently, it was demonstrated
367
that histone H2B proteins that were optimized for nu-
clear targeting could be used to reconstitute chromatin.
The histone/DNA cargo is condensed and protected from
degradation by nucleases that, together with the NLS,
ensure an efficient delivery of the DNA to the nucleus of
intact living mammalian cells (Pouton et al., 2007; Wag-
staff et al., 2008). In their study, they achieved an effi-
cient delivery of DNA to the nucleus of intact living
mammalian cells with a 6-fold higher level of transgene
expression than commercial liposomal delivery ap-
proaches (Wagstaff et al., 2008). This method, called
“chromofection,” resulted in the successful expression in
35% of cells to which DNA had been delivered, which is
40-fold more efficient than that observed for traditional
nonviral mediated techniques and 4- to 5-fold better
than previously engineered histone H2B monomers or
dimers (Pouton et al., 2007; Wagstaff et al., 2008). Chro-
mofection may represent an efficient means to deliver
large pieces of DNA (plasmid DNA of 6000 base pairs)
encoding multiple genes, with the potential to treat com-
plex genetic disorders (Wagstaff et al., 2008). In addition
to treating fatal monogenic diseases, the use of gene
therapy as an adjuvant treatment with radiotherapy
and chemotherapy has shown promising results. The
transfer of gene therapy from rare genetic conditions to
more common disorders, such as cancer or cardiovascu-
lar diseases is likely to benefit patients who will have a
wider choice of treatments available (Räty et al., 2008).
V. Applications for Therapy
A. Viral Therapy
Viruses have evolved to exploit the host’s cellular ma-
chinery to replicate their own genomes. For example,
adenoviruses target their genome to the cell nucleus by
a multistep process involving endocytosis, membrane
penetration, and cytoplasmic transport, and finally im-
port their DNA into the nucleus (Greber et al., 1996;
Leopold and Crystal, 2007).
Most current anti-human immunodeficiency virus
(HIV) therapies have focused on developing drugs that
inhibit viral entry, viral genome replication, and virus-
specific proteolysis. However, many viruses exploit cel-
lular kinases to facilitate subcellular targeting during
infection and to achieve specific phosphorylations of
their gene products (Alvisi et al., 2008). The kinase CK2
phosphorylates most viral proteins (Meggio and Pinna,
2003) by inducing an increase in the affinity between
importin and an NLS, thereby increasing nuclear pro-
tein import (Krosky et al., 2003; Marschall et al., 2003;
Alvisi et al., 2008). For example, the DNA replication of
the human cytomegalovirus (HCMV), a Herpesviridae
family member, depends on the DNA polymerase ho-
loenzyme, which is composed of a catalytic subunit
(pUL54) and a polymerase accessory protein or proces-
sivity factor (ppUL44) (Ertl and Powell, 1992; Mocarski
and Kemble, 1996; Alvisi et al., 2008). It has been re-
368 CHAHINE AND PIERCE
ported that, although the ppUL44-NLS is directly rec-
ognized by importin-␣/␤ for nuclear translocation, the
affinity of this interaction is strongly increased by the
presence of upstream sequences containing a site
(Ser413) for CK2 phosphorylation (Alvisi et al., 2008).
Thus, critical viral protein products such as ppUL44
exploit CK2 activity for proper intracellular localization.
This, in turn, implies the development of new antiviral
drugs directed against CK2 and may be a valuable di-
rection. Indeed, inhibition of the activity of these kinases
with cdk inhibitors, for example, blocks the replication of
many viruses including herpes simplex virus, HCMV,
varicella-zoster virus, Epstein-Barr virus, and HIV.
Thus, inhibiting kinases and specifically CK2 phosphor-
ylation may be effective, at least in part, through the
inhibition of protein transport into the nucleus (Alvisi et
al., 2008).
To prevent viral integration and transcription from
occurring, other compounds have been developed to in-
hibit the nuclear import of HIV-1 (Gasiorowski and
Dean. 2003). Nuclear translocation of HIV-1 preintegra-
tion complex (PIC) (a nucleic acid/protein complex) is
essential for viral replication, because it allows the PIC
to get into contact with the cellular chromatin (Haffar et
al., 2005). Arylene bis (methylketone) small-molecular-
weight compounds, such as the agent CNI-H1194, show
therapeutic promise by disrupting the formation of the
PIC, which could lead to inefficient nuclear import
(Dubrovsky et al., 1995; Popov et al., 1996; Gasiorowski
and Dean, 2003). However, there is still no consensus
about the mechanisms that govern nuclear import of the
HIV-1 PIC (Nisole and Saïb, 2004; Haffar et al., 2005).
Three HIV-1 proteins, matrix protein, integrase, and
viral protein R, have been proposed as karyophilic
agents that recruit the cellular nuclear import machin-
ery to the PIC (Haffar et al., 2005). However, the ability
of HIV-1 to transport its cDNA through an intact nu-
clear envelope depend on the karyophilic potential of
HIV-1 integrase via an importin-␣/␤-dependent mecha-
nism (Hearps and Jans, 2006). Integrase harbors multi-
ple NLSs that interact with importin-␣ and the impor-
tin-␣/␤ heterodimer (Hearps and Jans, 2006; Faustino et
al., 2007a). This classic nuclear import mechanism of
integrase highlights important potential therapeutic
targets for impeding the progression of HIV/AIDS. In-
deed, targeting matrix protein NLS-dependent nuclear
import by use of excess NLS mimetics has been shown to
inhibit HIV replication (Bukrinsky et al., 1993; Friedler
et al., 1998; Glushakova et al., 1999; Haffar et al., 2005).
For example, backbone cyclic peptides that have amino
acid sequences corresponding to the NLS of the HIV
matrix were able to inhibit nuclear import in in vitro
assay systems and HIV-1 replication in infected cultured
cells (Friedler et al., 1998). The NLS mimetics presum-
ably work by competing with the matrix protein NLS for
binding to importin-␣ (Haffar et al., 2005).
Arylene bis(methylketone) compounds have also been
described as a class of small-molecule inhibitors of HIV
nuclear import that directly target the matrix NLS
(Dubrovsky et al., 1995). For example, one of the com-
pounds of this group, the ITI-002, inactivates the NLS
by forming Schiff bases with lysine residues (through
the ketone groups) (Al-Abed et al., 2002), which can
neutralize the positive charges critical for NLS activity
(Haffar et al., 2005).
B. Cancer Therapy
The major obstacle that must be surmounted to de-
velop therapeutics that modify the general nuclear
transport machinery of cancer cells is the problem of
specificity for tumor cells versus normal cells (Kau et al.,
2004). To address this problem, researchers have used
the subcellular distribution of a mislocalized protein in
cancer cells to identify small molecules or peptide
aptamers that correctly redirect the proteins to the cor-
rect compartments or alter the cellular localization of a
protein (Kau et al., 2004). Analogous to monoclonal an-
tibodies, peptide aptamers are small proteins that con-
tain a structurally constrained variable region of ⬃20
amino acids, expressed as part of an inert scaffold such
as thioredoxin or green fluorescent protein (Colas et al.,
1996; Kau and Silver, 2003). The advantage of their
small size allows their structures to be solved and to
function inside cells (Colas et al., 1996). Some aptamers
have been designed to incorporate a NLS to translocate
their binding partner into the nucleus by use of the
nuclear transport machinery of the cell (Colas et al.,
2000; Kau and Silver, 2003). For example, peptide
aptamers, such as anti-Cdk2 and anti-Ste5 aptamers,
have been fused to an NLS as “transporters,” which
caused their targets to accumulate in the nucleus (Colas
et al., 2000). Such screening approaches could lead to the
discovery of novel compounds that have anticancer ac-
tivity, and compounds that provide new insights into the
role of nucleocytoplasmic trafficking and regulatory
pathways in cancer (Kau et al., 2004).
One of the major problems during cancer chemother-
apy is the development of resistance to cancer drugs.
Novel approaches have been developed by conjugating
an anticancer agent, such as a carboplatin analog, to a
poly(ethylene glycol) carrier and an NLS resulting in a
rapid internalization of the drug into M109 murine lung
carcinoma cells and an efficient accumulation in the
nucleus (Aronov et al., 2004). Although underused to-
day, use of an NLS to direct drugs to the nucleus may be
a novel and efficient way to improve drug efficacy in the
future.
A targeted radiotherapy of malignancies has been suc-
cessfully achieved through radiolabeled biomolecules,
such as peptides that recognize tumor-associated anti-
gens or growth-factor receptors (Goldenberg, 2002;
Reilly, 2005). However, these biomolecules have been
restricted almost exclusively to the cell surface. Novel
 THERAPEUTIC TARGETING OF NUCLEAR PROTEIN IMPORT IN DISEASE
strategies have recently emerged to overcome the deliv-
ery challenges through the conjugation of biomolecules
with NLSs to promote their internalization and routing
to the cell nucleus (Costantini et al., 2008). Most impor-
tantly, exciting new opportunities also have emerged by
use of NLS motifs to direct auger-electron-emitting ra-
diopharmaceuticals to the nucleus of cancer cells where
these electrons are highly potent in causing DNA strand
breaks, thereby killing the cells (Kassis, 2003). For ex-
ample, receptor tyrosine kinases and their cognate li-
gands are able to translocate to the nucleus through
NLS-mediated processes (Jans, 1994). In many types of
human malignancies, epidermal growth factor receptor
(EGFR) is overexpressed in nuclei from tumors in pa-
tients with breast and oropharyngeal carcinomas (Lo et
al., 2006). The internalization and nuclear translocation
of EGFR has been exploited by introducing auger-elec-
tron-emitting 111In-labeled EGF (111In-DTPA-hEGF) to
the nucleus of breast cancer cells (Chen et al., 2003;
Costantini et al., 2008). 111In and other auger-electron
emitters are highly cytotoxic and damaging to DNA
when they decay in close proximity to the cell nucleus.
This makes them highly selective for killing targeted
single-cancer cells. Indeed, Chen’s group found that
111
In-DTPA-hEGF possessed strong antitumor effects
(3-fold reduction in tumor growth) when administered to
athymic mice bearing EGFR-positive MDA-MB-468 hu-
man breast cancer xenografts (Chen et al., 2003; Costan-
tini et al., 2008).
Finally, an increasing number of oncogenes and tumor
suppressors have now been identified as important in-
tervention points for next-generation anticancer drugs.
In general, cellular p53 exists in low concentrations and
is relatively inactive, but during cellular stress, the
amount of p53 is increased and the protein is activated.
Depending on the cellular environment, activation of the
protein leads to either cell cycle arrest or apoptosis. The
network microtubules and the dynein motor proteins are
involved in facilitating p53 nuclear import (Pouton et al.,
2007). For example, treatment with the microtubule-
depolymerizing agent nocodazole reduces the extent of
nuclear p53 accumulation in vivo (Giannakakou et al.,
2000; Lam et al., 2002; Kau and Silver, 2003; Pouton et
al., 2007; Roth et al., 2007). This approach may also
improve the delivery of therapeutic DNA or drugs to the
nucleus in gene therapy or cancer therapy, because vi-
ruses and some proteins reach the perinuclear region by
following the microtubular network. However, because
of safety concerns associated with the use of viral vec-
tors, the development of safe, nonviral gene delivery
vectors is preferable (Pouton et al., 2007). For example,
Moffatt and colleagues (2006) designed a single vector
having multiple factors for both tumor-targeted and
nuclear-specific delivery, such as CNGRC/poly(ethylene
glycol)/polyethylenimine/DNA-p53/NLS/DNA nuclear tar-
geting signal. The CNGRC peptide was chosen as the tar-
geting ligand because of its high specificity for CD13,
369
which is expressed at extremely high levels in tumor cells
but not in the normal vasculature. This vector not only
targeted the nucleus of tumor cells but also the nucleus of
tumor-associated endothelial cells (Moffatt et al., 2006). In
conclusion, targeting proteins that are involved in nuclear
transport, in addition to the nuclear transport of factors
that have been associated with cancer, could prove to be a
powerful approach for controlling cancer cell growth.
Conclusions and Perspectives
Transport of macromolecules between the nucleus and
cytoplasm is a critical cellular process for eukaryotes,
and the machinery that mediates nucleocytoplasmic ex-
change is subject to multiple levels of control. This reg-
ulation is achieved by modulating the expression or
function of single cargoes, transport receptors, or the
transport channel itself. Each of these targets has an
increasingly broad impact on transport patterns and
capacity, and this hierarchy of control directly affects
gene expression, signal transduction, cell growth, and
disease. Naturally, if the import process is involved in
the pathogenesis of a disease, then it logically follows
that it may represent a potential therapeutic target.
Drug action and delivery can try to relocalize a protein
to alter its activity instead of simply blocking active
enzymatic sites (Gasiorowski and Dean, 2003). Introduc-
ing NLS peptides to compete with existing transporters
and inhibit protein movement into the nucleus can ef-
fectively deter growth processes. Alternatively, peptides
resembling NLS sequences can be used to target the
DNA toward the nucleus to facilitate nuclear uptake of
exogenous DNA. We are just beginning to realize the
potential of treating disease through targeting the nu-
clear import pathway. Increasing our understanding of
the factors that modulate nuclear protein import may
allow us to fully capitalize on the potential of using this
pathway to treat a great number of chronic diseases that
have augmented cell growth as a central feature.
Acknowledgments. This work was supported by the Canadian
Institutes for Health Research and through indirect research costs
provided by St Boniface Hospital and Research Foundation. M.N.C.
was supported by a Postdoctoral Fellowship from the Heart and
Stroke Foundation of Canada.
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